AccScience Publishing / IJB / Volume 12 / Issue 2 / DOI: 10.36922/IJB025500515
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RESEARCH ARTICLE

Development and assessment of dimethyl sulfoxide-free antifreeze gelatin methacryloyl hydrogels for integrated three-dimensional bioprinting and cryopreservation

Xin Li1,2,3,4 ,  Yukun Cao2,3,4 ,  Chengyuan Li1 ,  Chenxi Liu2 ,  Jia Tan2,5 ,  Xinli Zhou2,3,4* ,  Yang Yu6* ,  Xi Xia1*
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1 Department of Reproductive Medicine, Peking University Shenzhen Hospital, Shenzhen Peking University_the Hong Kong University of Science and Technology Medical Center, Shenzhen, Guangdong, China
2 Department of Biomedical Engineering, University of Shanghai for Science and Technology, Shanghai, China
3 Tumor Energy Therapy Laboratory, Shanghai Co-innovation Center for Energy Therapy of Tumors, Shanghai, China
4 Cryobiology Laboratory, Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai, China
5 Key Laboratory for Tissue Engineering of Jiangxi Province, School of Medical Information Engineering, Gannan Medical University, Ganzhou, Jiangxi, China
6 Department of Obstetrics and Gynecology, Peking University Third Hospital, Beijing, China
IJB 2026, 12(2), 025500515 https://doi.org/10.36922/IJB025500515
Received: 10 December 2025 | Accepted: 9 February 2026 | Published online: 20 February 2026
© 2026 by the Author(s).. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 International License ( https://creativecommons.org/licenses/by/4.0/ )
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Abstract

Three-dimensional (3D) bioprinting enables the fabrication of engineered tissues, but cell damage during printing and limitations in long-term preservation hinder practical applications. Traditional cryoprotectants, such as dimethyl sulfoxide (DMSO), introduce cytotoxicity and require complex removal, restricting immediate tissue usability. Here, we present an integrated extrusion-based bioprinting and DMSO-free antifreeze hydrogel strategy to produce cell-laden constructs with high post-thaw viability and proliferative capacity. Systematic optimization of bioink composition (6% L-proline with varying gelatin methacryloyl concentrations), extrusion parameters, and crosslinking conditions enabled high-fidelity scaffold fabrication while preserving cell viability and proliferation. Numerical simulations guided the maximum printable heights for fibers of different diameters, supporting construct scalability. Storing cell-laden 3D-printed scaffolds in cryovials at −80 °C effectively maintained high cell viability compared with alternative cooling protocols. Cells in 3D scaffolds exhibited superior post-thaw proliferation compared with two-dimensional culture, and the platform was validated using C2C12 myoblasts, achieving high survival and robust recovery of proliferative capacity. This study establishes a practical and versatile framework for integrating bioprinting and cryopreservation to support the generation of cell-laden constructs with preserved viability and structural integrity for regenerative medicine applications.

Graphical abstract
Keywords
Three-dimensional bioprinting
Cryopreservation
Gelatin methacryloyl
Antifreeze hydrogel
Dimethyl sulfoxide-free hydrogels
Printability optimization
Funding
This research was supported by the Sanming Project of Medicine in Shenzhen (No. SZSM202211043).
Conflict of interest
The authors declare no conflicts of interest.
References
  1. Liu K, Hu N, Yu Z, et al. 3D printing and bioprinting in urology. Int J Bioprint. 2023;9(6):0969. doi: 10.36922/ijb.0969

 

  1. Pantermehl S, Emmert S, Foth A, et al. 3D Printing for Soft Tissue Regeneration and Applications in Medicine. Biomedicines. 2021;9(4):336. doi: 10.3390/biomedicines9040336

 

  1. Wang H, Yang Y, Zhou X, et al. Rational design of mechanical bio-metamaterials for biomedical applications. Prog Mater Sci. 2026;156:101545. doi: 10.1016/j.pmatsci.2025.101545

 

  1. Habib A, Sathish V, Mallik S, et al. 3D Printability of Alginate-Carboxymethyl Cellulose Hydrogel. Materials. 2018;11(3):454. doi: 10.3390/ma11030454

 

  1. Emmermacher J, Spura D, Cziommer J, et al. Engineering considerations on extrusion-based bioprinting: interactions of material behavior, mechanical forces and cells in the printing needle. Biofabrication. 2020;12(2):025022. doi: 10.1088/1758-5090/ab7553

 

  1. Ning L, Guillemot A, Zhao J, et al. Influence of Flow Behavior of Alginate-Cell Suspensions on Cell Viability and Proliferation. Tissue Eng Part C Methods. 2016;22(7):652- 662. doi: 10.1089/ten.TEC.2016.0011

 

  1. Wu W, Xia R, Qian G, et al. Mechanostructures: Rational mechanical design, fabrication, performance evaluation, and industrial application of advanced structures. Prog Mater Sci. 2023;131:101021. doi: 10.1016/j.pmatsci.2022.101021

 

  1. Zhang YS, Haghiashtiani G, Hübscher T, et al. 3D extrusion bioprinting. Nat Rev Methods Primers. 2021;1(1):75. doi: 10.1038/s43586-021-00073-8

 

  1. Ng WL, Shkolnikov V. Optimizing cell deposition for inkjet-based bioprinting. Int J Bioprint. 2024;10(2):2135. doi: 10.36922/ijb.2135

 

  1. Law ACC, Wang R, Chung J, et al. Process parameter optimization for reproducible fabrication of layer porosity quality of 3D-printed tissue scaffold. J Intell Manuf. 2024;35(4):1825-1844. doi: 10.1007/s10845-023-02141-0

 

  1. Das S, Valoor R, Ratnayake P, et al. Low-concentration gelatin methacryloyl hydrogel with tunable 3D extrusion printability and cytocompatibility: exploring quantitative process science and biophysical properties. ACS Appl Bio Mater. 2024;7(5):2809-2835. doi: 10.1021/acsabm.3c01194

 

  1. Ji S, Guvendiren M. Recent Advances in Bioink Design for 3D Bioprinting of Tissues and Organs. Front Bioeng Biotechnol. 2017;5:23. doi: 10.3389/fbioe.2017.00023

 

  1. Malda J, Visser J, Melchels FP, et al. 25th anniversary article: Engineering hydrogels for biofabrication. Adv Mater. 2013;25(36):5011-5028. doi: 10.1002/adma.201302042

 

  1. Chang R, Nam J, Sun W. Effects of dispensing pressure and nozzle diameter on cell survival from solid freeform fabrication-based direct cell writing. Tissue Eng Part A. 2008;14(1):41-48. doi: 10.1089/ten.a.2007.0004

 

  1. Correia FP, Monteiro MV, Borralho M, et al. Advanced Toolboxes for Cryobioprinting Human Tissue Analogs. Adv Healthc Mater. 2025;14(10):2405011. doi: 10.1002/adhm.202405011

 

  1. Ziani K, Saenz-del-Burgo L, Pedraz J L, et al. Advances in Cryopreservation Strategies for 3D Biofabricated Constructs: From Hydrogels to Bioprinted Tissues. Int J Mol Sci. 2025;26(14):6908. doi: 10.3390/ijms26146908

 

  1. Ravanbakhsh H, Luo Z, Zhang X, et al. Freeform Cell-Laden Cryobioprinting for Shelf-Ready Tissue Fabrication and Storage. Matter. 2022;5(2):573-593. doi: 10.1016/j.matt.2021.11.020

 

  1. Liu M, Jiang S, Witman N, et al. Intrinsically cryopreservable, bacteriostatic, durable glycerohydrogel inks for 3D bioprinting. Matter. 2023;6(3):983-999. doi: 10.1016/j.matt.2022.12.013

 

  1. Liu M, Zhang X, Guo H, et al. Dimethyl Sulfoxide- Free Cryopreservation of Chondrocytes Based on Zwitterionic Molecule and Polymers. Biomacromolecules. 2019;20(10):3980-3988. doi: 10.1021/acs.biomac.9b01024

 

  1. Mahdavi SS, Abdekhodaie MJ, Kumar H, et al. Stereolithography 3D Bioprinting Method for Fabrication of Human Corneal Stroma Equivalent. Ann Biomed Eng. 2020;48(7):1955-1970. doi: 10.1007/s10439-020-02537-6

 

  1. Cao K, Shen L, Guo X, et al. Hydrogel Microfiber Encapsulation Enhances Cryopreservation of Human Red Blood Cells with Low Concentrations of Glycerol. Biopreserv Biobank. 2020;18(3):228-234. doi: 10.1089/bio.2020.0003

 

  1. Munesada D, Sakai D, Nakamura Y, et al. Investigation of the mitigation of DMSO-induced cytotoxicity by hyaluronic acid following cryopreservation of human nucleus pulposus cells. Int J Mol Sci. 2023;24(15):12289. doi: 10.3390/ijms241512289

 

  1. Pogozhykh D, Eicke D, Gryshkov O, et al. Towards reduction or substitution of cytotoxic dmso in biobanking of functional bioengineered megakaryocytes. Int J Mol Sci. 2020;21(20):7654. doi: 10.3390/ijms21207654

 

  1. Elliott GD, Wang S, Fuller BJ. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology. 2017;76:74-91. doi: 10.1016/j.cryobiol.2017.04.004

 

  1. Troitzsch RZ, Vass H, Hossack WJ, et al. Molecular mechanisms of cryoprotection in aqueous proline: light scattering and molecular dynamics simulations. J Phys Chem B. 2008;112(14):4290-4297. doi: 10.1021/jp076713m

 

  1. Szabados LSavouré A. Proline: a multifunctional amino acid. Trends Plant Sci. 2010;15(2):89-97. doi: 10.1016/j.tplants.2009.11.009

 

  1. Kaur G, Asthir B. Proline: a key player in plant abiotic stress tolerance. Biologia Plantarum. 2015;59(4):609-619. doi: 10.1007/s10535-015-0549-3

 

  1. Li X, Cao Y, Liu C, et al. l-Proline and GelMA hydrogel complex：An efficient antifreeze system for cell cryopreservation. Cryobiology. 2024;116:104942. doi: 10.1016/j.cryobiol.2024.104942

 

  1. Lee SC, Gillispie G, Prim P, et al. Physical and chemical factors influencing the printability of hydrogel-based extrusion bioinks. Chem Rev. 2020;120(19):10834-10886. doi: 10.1021/acs.chemrev.0c00015

 

  1. Hannah J, Zhou P. Regulation of DNA damage response pathways by the cullin-RING ubiquitin ligases. DNA Repair. 2009;8(4):536-543. doi: 10.1016/j.dnarep.2009.01.011

 

  1. Yue K, Trujillo-de Santiago G, Alvarez MM, et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials. 2015;73:254-271. doi: 10.1016/j.biomaterials.2015.08.045

 

  1. Paxton N, Smolan W, Böck T, et al. Proposal to assess printability of bioinks for extrusion-based bioprinting and evaluation of rheological properties governing bioprintability. Biofabrication. 2017;9(4):044107. doi: 10.1088/1758-5090/aa8dd8

 

  1. Herrada-Manchón H, Fernández M A, Aguilar E. Essential guide to hydrogel rheology in extrusion 3D printing: how to measure it and why it matters? Gels. 2023;9(7):517. doi: 10.3390/gels9070517

 

  1. Zhang D, Liu J, Liu X, et al. Biomechanical and mechanobiological design for bioprinting functional microvasculature. Appl Phys Rev. 2025;12(1):011332. doi: 10.1063/5.0227692

 

  1. Kielbassa C, Epe B. DNA damage induced by ultraviolet and visible light and its wavelength dependence. Methods Enzymol. 2000;319:436-445. doi: 10.1016/s0076-6879(00)19041-x

 

  1. Elango JZamora-Ledezma C. Rheological, structural, and biological trade-offs in bioink design for 3d bioprinting. Gels. 2025;11(8):659. doi: 10.3390/gels11080659

 

  1. Nair K, Gandhi M, Khalil S, et al. Characterization of cell viability during bioprinting processes. Biotechnol J. 2009;4(8):1168-1177. doi: 10.1002/biot.200900004

 

  1. Blaeser A, Duarte Campos DF, Puster U, et al. Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity. Adv Healthc Mater. 2016;5(3):326-333. doi: 10.1002/adhm.201500677

 

  1. Xie M, Gao Q, Zhao H, et al. Electro-Assisted Bioprinting of Low-Concentration GelMA Microdroplets. Small. 2019;15(4):e1804216. doi: 10.1002/smll.201804216

 

  1. Adhikari J, Roy A, Das A, et al. Effects of Processing Parameters of 3D Bioprinting on the Cellular Activity of Bioinks. Macromol Biosci. 2021;21(1):e2000179. doi: 10.1002/mabi.202000179

 

  1. Zhang Y, O’Mahony A, He Y, et al. Hydrodynamic shear stress’ impact on mammalian cell properties and its applications in 3D bioprinting. Biofabrication. 2024;16(2):022003. doi: 10.1088/1758-5090/ad22ee

 

  1. Chang SF, Chang CA, Lee DY, et al. Tumor cell cycle arrest induced by shear stress: Roles of integrins and Smad. Proc Natl Acad Sci USA. 2008;105(10):3927-3932. doi: 10.1073/pnas.0712353105

 

  1. Ahn G, Min KH, Kim C, et al. Precise stacking of decellularized extracellular matrix based 3D cell-laden constructs by a 3D cell printing system equipped with heating modules. Sci Rep. 2017;7(1):8624. doi: 10.1038/s41598-017-09201-5

 

  1. Cadet J, Douki T, Ravanat JL. Oxidatively generated damage to cellular DNA by UVB and UVA radiation. Photochem Photobiol. 2015;91(1):140-155. doi: 10.1111/php.12368

 

  1. Mazur P. Cryobiology: the freezing of biological systems. Science. 1970;168(3934):939-949. doi: 10.1126/science.168.3934.939

 

  1. Yu M, Marquez-Curtis L, Elliott JAW. Cryopreservation-induced delayed injury and cell-type-specific responses during the cryopreservation of endothelial cell monolayers. Cryobiology. 2024;115:104857. doi: 10.1016/j.cryobiol.2024.104857

 

  1. Best B P. Cryoprotectant Toxicity: Facts, Issues, and Questions. Rejuvenation Res. 2015;18(5):422-436. doi: 10.1089/rej.2014.1656
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International Journal of Bioprinting, Electronic ISSN: 2424-8002 Print ISSN: 2424-7723, Published by AccScience Publishing
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